MicroRNA-661 Suppresses Proliferation and Migration of Pulmonary Artery Smooth Muscle Cells and Endothelial Cells by Targeting CCND2.

Introduction: Pulmonary arterial hypertension (PAH) is a fatal cardiovascular disorder driven by pulmonary vascular remodeling, with high morbidity and mortality. Current therapies only alleviate symptoms and cannot reverse disease progression. Our previous study has demonstrated that microRNA-661 (miR-661) inhibits Human pulmonary artery smooth muscle cell (HPASMC) and Human pulmonary artery endothelial cell (HPAEC) proliferation, but its underlying molecular mechanism in PAH remains unclear. This study aimed to identify the target gene of miR-661 and elucidate its regulatory role in PAH pathogenesis, seeking novel therapeutic targets for this incurable disease. Methods: Serum samples from 12 PAH patients and 12 healthy volunteers were enrolled this research. Human pulmonary artery smooth muscle cells (HPASMCs) and endothelial cells (HPAECs) were cultured under normoxic or hypoxic conditions. MiR-661 mimics/inhibitors and CCND2 plasmids were transfected into cells. RNA-seq screened differentially expressed genes, and dual-luciferase reporter assay verified the target binding of miR-661. RT-qPCR and Western blot detected gene/protein expression, while MTT, EdU, Transwell and flow cytometry assessed cell proliferation, migration and cycle progression. Results: We perform RNA-seq and luciferase assay and found that CCND2 is a direct miR-661 target that is repressed via 3'UTR binding. miR-661 is lower expressed while the expression of CCND2 is higher in PAH patient serum and hypoxic HPASMCs/HPAECs, showing a significant negative correlation. CCND2 overexpression promoted pulmonary artery cell cycle progression, proliferation and migration under hypoxia, while miR-661 overexpression reversed these effects. In addition, we found that Stattic, a specific inhibitor of STAT3 (an upstream regulatory protein of CCND2), could inhibit HPASMC cell proliferation and migration and this effect is enhanced by miR-661. Discussion: This study clarifies that miR-661 suppresses HPASMC and HPAEC proliferation or migration by targeting CCND2, and the miR-661/CCND2 axis is a key regulatory pathway in PAH-related vascular remodeling. Hypoxia-induced miR-661 downregulation leads to CCND2 accumulation, driving abnormal pulmonary artery cell biological behaviors. miR-661/CCND2 axis regulates PAH progression, providing a potential therapeutic target.